Abstract 838: microRNA-210 Regulates Endothelial Cell Response to Hypoxia
Background. MicroRNAs (miRNAs) are small non-protein-coding RNAs that downregulate gene expression by inhibiting mRNA translation in mammalian cells. In this study, it was investigated miRNAs regulation and functional role in endothelial cell response to hypoxia.
Methods and results. Human Umbelical Vein Endothelial Cells (HUVEC) were cultured either in normoxic conditions or in the presence of 1% oxygen for 8 – 48 hours. Then, a selection of 157 miRNAs was measured using a real-time PCR assay designed to quantify only mature miRNAs. The expression of miR-210 and miR-150 progressively increased upon exposure to hypoxia. At 48 hours, miR-210 and miR-150 were 35±5 and 14±4 fold more expressed than control (p<0.001 and <0.007, respectively). Both responses were independent of cell confluency and were not limited to vascular cells. In additional experiments it was characterized miR-210 regulation and its the role on HUVEC function. Neither acidification nor oxidative stress, two components of the cell response to hypoxia, modulated miR-210. Further, miR-210 overexpression in normoxic HUVEC stimulated the formation of capillary-like structures on Matrigel and VEGF-driven cell migration. Conversely, miR-210 blockade via LNA-antimiR transfection inhibited the formation of capillary-like structures stimulated by hypoxia and decreased cell migration in response to VEGF. miR-210 overexpression did not affect HUVEC growth in both normoxia and hypoxia. Conversely, antimiR-210 transfection inhibited cell growth both in normoxia (66%±8 of control; p<0.003) and in hypoxia (50%±9 of control; p<0.001). While DNA synthesis was not affected, miR-210 blockade induced apoptosis, as assessed by measuring cytoplasmic nucleosomal DNA. One relevant target of miR-210 in hypoxia was Ephrin-A3, since miR-210 was necessary to downmodulate its expression upon hypoxia. Moreover, miR-210 overexpression in normoxic condition was sufficient to inhibit Ephrin-A3 protein level. Finally, luciferase reporter assays showed that Ephrin-A3 was a direct target of miR-210.
Conclusions. miR-210 upregulation is a crucial element of cell response to hypoxia, affecting cell survival, migration and differentiation. #